Complete Biochemical Characterization of Pantaphos Biosynthesis Highlights an Unusual Role for a SAM‐Dependent Methyltransferase

Pantaphos is small molecule virulence factor made by the plant pathogen Pantoea ananatis. An 11 gene operon, designated hvr for high virulence, is required for production of this phosphonic acid natural product, but the metabolic steps used in its production have yet to be established. Herein, we determine the complete biosynthetic pathway using a combination of bioinformatics, in vitro biochemistry and in vivo heterologous expression. Only 6 of the 11 hvr genes are needed to produce pantaphos, while a seventh is likely to be required for export. Surprisingly, the pathway involves a series of O‐methylated intermediates, which are then hydrolyzed to produce the final product. The methylated intermediates are produced by an irreversible S‐adenosylmethione (SAM)‐dependent methyltransferase that is required to drive a thermodynamically unfavorable dehydration in the preceding step, a function not previously attributed to members of this enzyme class. Methylation of pantaphos by the same enzyme is also likely to limit its toxicity in the producing organism. The pathway also involves a novel flavin‐dependent monooxygenase that differs from homologous proteins due to its endogenous flavin‐reductase activity. Heterologous production of pantaphos by Escherichia coli strains expressing the minimal gene set strongly supports the in vitro biochemical data. Biosynthesis of the phosphonate natural product pantaphos involves a SAM‐dependent methylation reaction, despite the fact that no SAM‐derived carbon atoms are present in the final product. This irreversible reaction provides the driving force needed to overcome the unfavorable thermodynamics of the preceding dehydration, while reducing the toxicity of the biosynthetic intermediates.